PROJECT SUMMARY Significance: Translation initiation is a highly regulated step in eukaryotic gene expression and its dysregulation is linked to heritable human diseases and cancer. Translational regulation depends on cellular condition-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. A predictive understanding of these mRNA characteristics is needed to harness the broad therapeutic potential of drugs that target translation factors, develop new translation-based therapies, and engineer therapeutic mRNAs. Approach: Our work aims to comprehensively identify 5?-UTR sequences that enhance or repress translation and illuminate their underlying mechanisms. We hypothesize that 5?-UTRs with unexplained high or low translation activity bind proteins that function as mRNA-specific translational activators or repressors. We envision two broad categories of translational enhancers: 5?-UTR sequences or RNA structures that bind to core initiation factors preferentially, and 5?-UTR elements that bind novel factors whose roles in ribosome recruitment remain to be elucidated. In support of the first model, we have recently demonstrated strong sequence preferences for yeast eIF4G1 and found that its preferred binding motif, oligo-uridine, occurs naturally in the 5?-UTRs of hundreds of genes and enhances their translation. Aim 1 will leverage a new method developed in our laboratory that allows highly parallel dissection of candidate cis regulatory elements to define the contribution of specific 5?-UTR features to ribosome recruitment activity in cell lysates. We use S. cerevisiae (budding yeast) because 5?- UTR isoforms are experimentally well defined in this organism and we can exploit a wealth of genetic, biochemical and structural data. Aim 2 will reveal how much of the observed variance in ribosome recruitment is directly explained by differences in affinities for core initiation factors. Aim 3 will identify novel factors that bind 5?-UTR enhancer and silencer elements using crosslinking and mass spectrometry; investigate their mechanisms that alter ribosome recruitment in vitro; and explore their impact on regulated translation in vivo. Together, this work will reveal how differences in mRNA primary sequence lead to large and regulated differences in translation. Because the eukaryotic translational machinery and regulatory paradigms are highly conserved, the molecular insights gained here are likely to be broadly illuminating for understanding translational control, including in pathophysiological processes in humans.